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Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Surface plasmon resonance (SPR) proves to be one of the most effective methods of label-free detection and has been integral for the study of biomolecular interactions and the development of biosensors. This trend delves into the latest SPR research and progress built upon the Kretschmann configuration, a pivotal platform, and highlights three key developments that have enhanced the capabilities of the technique. We will first cover a range of explorations of novel plasmonic materials that have shaped SPR performance. Innovative signal transduction and collection, which leverages traditional materials and emerging alternatives, will then be discussed. Finally, the evolving landscape of data analysis, including the integration of machine learning algorithms to navigate complex SPR datasets, will be reviewed. We will also discuss the implementation of these improvements that have enabled new biosensing functions. These advancements not only pave the way for enhanced biosensing in general but also open new avenues for the technique to play a more significant role in research concerning human health., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

A majority of biomimetic membranes used for current biophysical studies rely on planar structures such as supported lipid bilayer (SLB) and self-assembled monolayers (SAMs). While they have facilitated key information collection, the lack of curvature makes these models less effective for the investigation of curvature-dependent protein binding. Here, we report the development and characterization of curved membrane mimics on a solid substrate with tunable curvature and ease in incorporation of cellular membrane components for the study of protein-membrane interactions. The curved membranes were generated with an underlayer lipid membrane composed of DGS-Ni-NTA and POPC lipids on the substrate, followed by the attachment of histidine-tagged cholera toxin (his-CT) as a capture layer. Lipid vesicles containing different compositions of gangliosides, including GA 1 , GM 1 , GT 1b , and GQ 1b , were anchored to the capture layer, providing fixation of the curved membranes with intact structures. Characterization of the curved membrane was accomplished with surface plasmon resonance (SPR), fluorescence recovery after photobleaching (FRAP), and nano-tracking analysis (NTA). Further optimization of the interface was achieved through principal component analysis (PCA) to understand the effect of ganglioside type, percentage, and vesicle dimensions on their interactions with proteins. In addition, Monte Carlo simulations were employed to predict the distribution of the gangliosides and interaction patterns with single point and multipoint binding models. This work provides a reliable approach to generate robust, component-tuning, and curved membranes for investigating protein interactions more pertinently than what a traditional planar membrane offers.

Phosphoinositide 3-kinase α (PIK3CA) is one of the most mutated genes across cancers, especially breast, gynecologic, and head and neck squamous cell carcinoma tumors. Mutations occur throughout the gene, but hotspot mutations in the helical and kinase domains predominate. The therapeutic benefit of isoform-selective PI3Kα inhibition was established with alpelisib, which displays equipotent activity against the wild-type and mutant enzyme. Inhibition of wild-type PI3Kα is associated with severe hyperglycemia and rash, which limits alpelisib use and suggests that selectively targeting mutant PI3Kα could reduce toxicity and improve efficacy. Here we describe STX-478, an allosteric PI3Kα inhibitor that selectively targets prevalent PI3Kα helical- and kinase-domain mutant tumors. STX-478 demonstrated robust efficacy in human tumor xenografts without causing the metabolic dysfunction observed with alpelisib. Combining STX-478 with fulvestrant and/or cyclin-dependent kinase 4/6 inhibitors was well tolerated and provided robust and durable tumor regression in ER+HER2- xenograft tumor models., Significance: These preclinical data demonstrate that the mutant-selective, allosteric PI3Kα inhibitor STX-478 provides robust efficacy while avoiding the metabolic dysfunction associated with the nonselective inhibitor alpelisib. Our results support the ongoing clinical evaluation of STX-478 in PI3Kα-mutated cancers, which is expected to expand the therapeutic window and mitigate counterregulatory insulin release. See related commentary by Kearney and Vasan, p. 2313. This article is featured in Selected Articles from This Issue, p. 2293., (©2023 The Authors; Published by the American Association for Cancer Research.)

Small-molecule drugs have enabled the practice of precision oncology for genetically defined patient populations since the first approval of imatinib in 2001. Scientific and technology advances over this 20-year period have driven the evolution of cancer biology, medicinal chemistry, and data science. Collectively, these advances provide tools to more consistently design best-in-class small-molecule drugs against known, previously undruggable, and novel cancer targets. The integration of these tools and their customization in the hands of skilled drug hunters will be necessary to enable the discovery of transformational therapies for patients across a wider spectrum of cancers., Significance: Target-centric small-molecule drug discovery necessitates the consideration of multiple approaches to identify chemical matter that can be optimized into drug candidates. To do this successfully and consistently, drug hunters require a comprehensive toolbox to avoid following the "law of instrument" or Maslow's hammer concept where only one tool is applied regardless of the requirements of the task. Combining our ever-increasing understanding of cancer and cancer targets with the technological advances in drug discovery described below will accelerate the next generation of small-molecule drugs in oncology., (©2023 The Authors; Published by the American Association for Cancer Research.)

SARS-CoV-2 has been shown to enter and infect human cells via interactions between spike protein (S glycoprotein) and angiotensin-converting enzyme 2 (ACE2). As such, it may be possible to suppress the infection of the virus via the blocking of this binding interaction through the use of specific peptides that can mimic the human ACE 2 peptidase domain (PD) α 1-helix. Herein, we report the use of competitive assays along with surface plasmon resonance (SPR) to investigate the effect of peptide sequence and length on spike protein inhibition. The characterization of these binding interactions helps us understand the mechanisms behind peptide-based viral blockage and develop SPR methodologies to quickly screen disease inhibitors. This work not only helps further our understanding of the important biological interactions involved in viral inhibition but will also aid in future studies that focus on the development of therapeutics and drug options. Two peptides of different sequence lengths, [30-42] and [22-44], based on the α 1-helix of ACE2 PD were selected for this fundamental investigation. In addition to characterizing their inhibitory behavior, we also identified the critical amino acid residues of the RBD/ACE2-derived peptides by combining experimental results and molecular docking modeling. While both investigated peptides were found to effectively block the RBD residues known to bind to ACE2 PD, our investigation showed that the shorter peptide was able to reach a maximal inhibition at lower concentrations. These inhibition results matched with molecular docking models and indicated that peptide length and composition are key in the development of an effective peptide for inhibiting biophysical interactions. The work presented here emphasizes the importance of inhibition screening and modeling, as longer peptides are not always more effective.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)-based lipid profiling is a powerful method to study the cytotoxicity of chemical exposure to microorganisms at the single cell level. We report here a combined approach of machine learning (ML) and microchip-based MALDI-time of flight (TOF) mass spectrometry to investigate the cytotoxic effect of herbicides on algae through single cell lipid profiling. Algal species Selenastrum capricornutum was chosen as the target system, and its exposure to different doses of common chemical herbicides and the resulting cytotoxic behaviors under various stress conditions were characterized. A lipid library for S. capricornutum has been established with 63 identified lipids that include glycosyldiacylglycerols and triacylglycerols. We demonstrated that major alternations occurred for lipids with functional groups of digalactosyldiacylglycerol (DGDG), triacylglycerol (TAG), and monogalactosyldiacylglycerol (MGDG). DGDG was shown to decrease upon exposure to herbicides of norflurazon and atrazine, while some MGDG and TAG lipids would increase for norflurazon. Compared to other algae, S. capricornutum was more strongly impacted by norflurazon than atrazine while the latter was observed to have a greater effect on C. reinhardtii . Machine learning algorithms have been applied to improve the classification of herbicide impact and help identify lipid species affected by the chemical exposure. A total of 69 machine learning models were trained and tested for the identification of ideal algorithms in the classification process, in which flexible discriminant analysis and support vector machine model were found to be the most accurate and consistent. The ML algorithms accurately differentiated herbicide impact and have identified cytotoxic differences that were previously hidden. The results suggest that herbicides express toxicity among different algae likely on the basis of metabolic differences. The ML-assisted method proves to be highly effective and can provide an advanced technological platform for probing cytotoxicity for bacterial species and in metabolic pathway analysis.

Misuse of agrochemicals has a long-lasting negative impact on aquatic systems. Mismanagement of herbicides in agri-food sectors is often linked to a simultaneous decline in the health of downstream waterways. However, monitoring the herbicide levels in these areas is a laborious task, and modern analytical approaches, such as solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) and enzyme-linked immunosorbent assay, are low-throughput and require significant sample preparation. We report here the use of microchip technology in combination with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for the assessment of the ecotoxicological effect of agrochemicals on aquatic species at the single-cell level. This approach quantifies the fluctuations in lipid content in sentinel organisms and targets the microalga, Chlamydomonas reinhardtii ( C. reinhardtii ), as the model system. Specifically, we investigated the cytotoxicity of three herbicides (atrazine, clomazone, and norflurazon) on C. reinhardtii by analyzing the lipid component variation upon assorted herbicide exposure. Lipidomic profiling reveals a significantly altered lipid content at >EC 50 in atrazine-exposed cells. The response for norflurazon showed similar trends but diminished in magnitude, while the result for clomazone was near muted. At lower herbicide concentrations, digalactosyldiacylglycerols showed a rapid decrease in abundance, while several other lipids displayed a moderate increase. The microchip-based MALDI technique demonstrates the ability to achieve lipidomic profiling of aquatic species exposed to different stressors, proving effective for high-throughput screening and single-cell analysis in ecotoxicity studies.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. IMPLICATIONS: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing., (©2021 American Association for Cancer Research.)

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254., Experimental Design: LXH254 was profiled in biochemical, in vitro , and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF., Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo , LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive., Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF., (©2020 American Association for Cancer Research.)

Multiple sclerosis (MS) is an autoimmune disease that damages the myelin sheaths of nerve cells in the central nervous system. An individual suffering from MS produces increased levels of antibodies that target cell membrane components, such as phospholipids, gangliosides, and membrane proteins. Among them, anti-ganglioside antibodies are considered as important biomarkers to differentiate MS from other diseases that exhibit similar symptoms. We report here a label-free method for detecting a series of antibodies against gangliosides in serum by surface plasmon resonance imaging (SPRi) in combination with a carbohydrate microarray. The ganglioside array was fabricated with a plasmonically tuned, background-free biochip, and coated with a perfluorodecyltrichlorosilane (PFDTS) layer for antigen attachment as a self-assembled pseudo-myelin sheath. The chip was characterized with AFM and matrix-assisted laser desorption ionization mass spectrometry, demonstrating effective functionalization of the surface. SPRi measurements of patients' mimicking blood samples were conducted. A multiplexed detection of antibodies for anti-GT 1b , anti-GM 1 , and anti-GA 1 in serum was demonstrated, with a working range of 1 to 100 ng/mL, suggesting that it is well suited for clinical assessment of antibody abnormality in MS patients. Statistical analyses, including PLS-DA and PCA show the array allows comprehensive characterization of cross reactivity patterns between the MS specific antibodies and can generate a wide range of information compared to traditional end point assays. This work uses PFDTS surface functionalization and enables direct MS biomarker detection in serum, offering a powerful alternative for MS assessment and potentially improved patient care.

Single cell lipid profiling is a powerful tool to connect membrane composition and its changes within individual cells to specific biochemical functions or stimuli, but current approaches are inadequate due to the complex nature of the cells and technical limitation in analysis. Herein we report a new method with plasmonic substrates capable of cell localization and enhanced lipid ionization through thin-gold-film MALDI-MS. We performed lipidomic profiling of algae single cells with a 120-well microarray and identified more than 50 lipids in C. reinhardtii without an extraction process. The substrate was used for probing toxicological effect of herbicide atrazine on the algae's lipidome, demonstrating molecular changes in glycerol lipid profiles. Fast location of cells with metal-enhanced fluorescence (MEF) and subsequent precise and direct ionization of the LDI process contribute to the enhanced performance, allowing for assessment of lipid changes concurrent with atrazine affected populations. This method that combines microarrays, MEF, and MALDI-MS presents an effective platform for lipidomic study of single cells and for environmental toxicity study with microorganisms.

KRAS , an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro , sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo , suggesting a MAPK pathway-dependent mechanism and possible non-MAPK pathway-dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRAS G12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors., (©2019 American Association for Cancer Research.)

Inhibitors targeting BRAF and its downstream kinase MEK produce robust response in patients with advanced BRAF V600 -mutant melanoma. However, the duration and depth of response vary significantly between patients; therefore, predicting response a priori remains a significant challenge. Here, we utilized the Novartis collection of patient-derived xenografts to characterize transcriptional alterations elicited by BRAF and MEK inhibitors in vivo , in an effort to identify mechanisms governing differential response to MAPK inhibition. We show that the expression of an MITF -high, "epithelial-like" transcriptional program is associated with reduced sensitivity and adaptive response to BRAF and MEK inhibitor treatment. On the other hand, xenograft models that express an MAPK-driven "mesenchymal-like" transcriptional program are preferentially sensitive to MAPK inhibition. These gene-expression programs are somewhat similar to the MITF -high and -low phenotypes described in cancer cell lines, but demonstrate an inverse relationship with drug response. This suggests a discrepancy between in vitro and in vivo experimental systems that warrants future investigations. Finally, BRAF V600 -mutant melanoma relies on either MAPK or alternative pathways for survival under BRAF and MEK inhibition in vivo , which in turn predicts their response to further pathway suppression using a combination of BRAF , MEK, and ERK inhibitors. Our findings highlight the intertumor heterogeneity in BRAF V600 -mutant melanoma, and the need for precision medicine strategies to target this aggressive cancer., (©2019 American Association for Cancer Research.)

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAF V600 -mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAF V600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers., (©2018 American Association for Cancer Research.)

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAF V600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors. Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537-48. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated BRAF -mutant melanoma. Experimental Design: The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50-700 mg) or twice-daily (75-150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic BRAF -mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib. Results: Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of BRAF V600E-mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar-plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4-not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9-3.7). Conclusions: Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. Clin Cancer Res; 23(18); 5339-48. ©2017 AACR ., (©2017 American Association for Cancer Research.)

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

The discovery of a highly potent and selective small molecule inhibitor 9 for in vitro target validation of MNK1/2 kinases is described. The aminopyrazine benzimidazole series was derived from an HTS hit and optimized by utilization of a docking model, conformation analysis, and binding pocket comparison against antitargets.

Despite the clinical success of RAF inhibitors in BRAF-mutated melanomas, attempts to target RAF kinases in the context of RAS-driven or otherwise RAF wild-type tumours have not only been ineffective, but RAF inhibitors appear to aggravate tumorigenesis in these settings. Subsequent preclinical investigation has revealed several regulatory mechanisms, feedback pathways and unexpected enzymatic quirks in the MAPK pathway, which may explain this paradox. In this review, we cover the various proposed molecular mechanisms for the RAF paradox, the clinical consequences and strategies to overcome it.

The RAS-RAF-MEK (MAP-ERK kinase)-ERK (extracellular signal-regulated kinase) pathway plays a central role in driving proliferation, survival, and metastasis signals in tumor cells, and the prevalence of oncogenic mutations in RAS and BRAF and upstream nodes makes this pathway the focus of significant oncology drug development efforts. This focus has been justified by the recent success of BRAF and MEK inhibitors in prolonging the lives of patients with BRAF(V600E/K)-mutant melanoma. Although it is disappointing that cures are relatively rare, this should not detract from the value of these agents to patients with cancer and the opportunity they provide in allowing us to gain a deeper understanding of drug response and resistance. These insights have already provided the basis for the evaluation of alternative dosing regimens and combination therapies in patients with melanoma., (©2014 AACR)

The RAS-RAF-MEK-ERK pathway is a key driver of proliferation and survival signals in tumor cells and has been the focus of intense drug development efforts over the past 20 years. The recent regulatory approval of RAF inhibitors and a MAP-ERK kinase (MEK) inhibitor for metastatic melanoma provides clinical validation of tumor dependency on this pathway. Unfortunately, the therapeutic benefit of these agents is often short lived and resistance develops within a matter of months. Preclinical models of resistance to vemurafenib have provided critical insights into predicting, validating, and characterizing potential mechanisms. A key observation has been that vemurafenib-resistant tumor cells suffer a fitness deficit in the absence of drug treatment and this led to the predication that modulating the selective pressure of drug treatment through intermittent dosing could delay or prevent the emergence of resistant tumors. Most importantly, the preclinical data are supported by observations in vemurafenib-treated patients with melanoma providing a strong rationale for clinical testing of alternative dosing regimens., (©2013 AACR.)

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain - insights that would be difficult or impossible to extract by use of radiolabeled compounds.

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with ≥50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)→MEK→ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.

Purpose: The purpose of this preclinical study was to determine the effectiveness of RAF265, a multikinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response., Experimental Design: Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated., Results: Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAF(V600E/K)), whereas eight of 17 (47%) tumors were BRAF wild type (BRAF(WT)). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAF(WT), of which 1 carried c-KIT(L576P) and another N-RAS(Q61R) mutation, while only 2 (29%) of the responding tumors were BRAF(V600E/K). Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11., Conclusions: Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles., (©2012 AACR.)

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo. Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung. We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle. The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN. The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice. The majority of the asODN was cleared from blood, with a half-life of >10 hours compared with <1 hour for free asODN. When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein. No inhibition was found for nontargeted formulations or for free asODN. A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN.

Antisense oligodeoxynucleotides (asODN) are therapeutic agents that are designed to inhibit the expression of disease-related genes. However, their therapeutic use may be hindered due to their rapid clearance from blood and their inefficiency at crossing cell membranes. Cationic liposome complexes have been used to enhance the intracellular delivery of asODN in vitro; however, this type of carrier has unfavorable pharmacokinetics for most in vivo applications. Significant therapeutic activity of cationic liposomal asODN following systemic administration has not been demonstrated. In an effort to develop improved liposomal carriers for asODN for in vivo applications, we have evaluated the physical characteristics of two formulations which represent alternatives to cationic liposome-asODN complexes: asODN passively entrapped within neutral liposomes (PELA) and asODN formulated in a novel coated cationic liposomal formulation (CCL). Our results confirm that PELA can be extruded to small diameters that are suitable for intravenous administration. PELA are stable in human plasma; however, the incorporation efficiency is relatively low ( approximately 20%). The CCL formulation can also be extruded to small diameters (<200 nm), with significantly higher (80-100%) incorporation efficiency and are stable in 50% human plasma at 37 degrees C. A liposomal carrier for asODN with these characteristics may provide a significant therapeutic advantage over free asODN for some therapeutic applications.

Background: Advanced-stage neuroblastoma resists conventional treatment; hence, novel therapeutic approaches are required. We evaluated the use of c-myb antisense oligodeoxynucleotides (asODNs) delivered to cells via targeted immunoliposomes to inhibit c-Myb protein expression and neuroblastoma cell proliferation in vitro., Methods: Phosphorothioate asODNs and control sequences were encapsulated in cationic lipid, and the resulting particles were coated with neutral lipids to produce coated cationic liposomes (CCLs). Monoclonal antibodies directed against the disialoganglioside GD(2) were covalently coupled to the CCLs. (3)H-labeled liposomes were used to measure cellular binding, and cellular uptake of asODNs was evaluated by dot-blot analysis. Growth inhibition was quantified by counting trypan blue dye-stained cells. Expression of c-Myb protein was examined by western blot analysis., Results: Our methods produced GD(2)-targeted liposomes that stably entrapped 80%-90% of added c-myb asODNs. These liposomes showed concentration-dependent binding to GD(2)-positive neuroblastoma cells that could be blocked by soluble anti-GD(2) monoclonal antibodies. GD(2)-targeted liposomes increased the uptake of asODNs by neuroblastoma cells by a factor of fourfold to 10-fold over that obtained with free asODNs. Neuroblastoma cell proliferation was inhibited to a greater extent by GD(2)-targeted liposomes containing c-myb asODNs than by nontargeted liposomes or free asODNs. GD(2)-targeted liposomes containing c-myb asODNs specifically reduced expression of c-Myb protein by neuroblastoma cells. Enhanced liposome binding and asODN uptake, as well as the antiproliferative effect, were not evident in GD(2)-negative cells., Conclusions: Encapsulation of asODNs into immunoliposomes appears to enhance their toxicity toward targeted cells while shielding nontargeted cells from antisense effects and may be efficacious for the delivery of drugs with broad therapeutic applications to tumor cells.

Past studies in our laboratory have shown that low levels of UVB can cause changes in the optical properties of organ cultured ocular lenses, while other research has shown that in vitro UV radiation causes decreases in leucine aminopeptidase activity in homogenates of crystalline lens material. Therefore we have investigated whether there is a relationship between such decreases in enzyme activity and changes in lens optics and structure. Organ cultured bovine lenses were irradiated with low doses of UVB, and lens optics, histology and leucine aminopeptidase activity (leucine beta-naphthylamide hydrolysis at pH 7.5) were assessed daily. Lenses irradiated with 0.1 J cm-2 UVB showed a decrease of about 30% in leucine aminopeptidase activity 1 h after irradiation, while changes in lens optics were not observed until at least 24 h after irradiation. Histological examination of the lens anterior epithelium revealed changes in epithelial cells ranging from pyknotic nuclei to large areas of cell fragmentation. The results of this study suggest that a decrease in soluble aminopeptidase activity in lens epithelial cells may be a direct result of the epithelial cell damage rather than an effect of UVB on the enzyme per se.

The effects of repeated exposures to UV-A (335 nm) and UV-B (305 nm) radiation on the crystalline lens were studied by treating cultured bovine lenses daily or weekly. The effects of irradiation on lens optical quality were monitored using an automated scanning laser system that records both relative transmittance and focal length across the lens. Relatively low radiant exposures of UV-B were used (0.06, 0.03, 0.01 J/cm2) compared to UV-A (1.44 J/cm2). In total, 38 treated lenses and 32 controls were cultured for times ranging from 400-1000 hours. Results indicate that this range of UV-B exposure may represent the threshold for in vitro UV-B induced opacification. Lenses treated weekly with 0.06 J/cm2 UV-B showed a significant decrease in transmittance compared to controls 69 hours after the first treatment and an increase in focal length variability. The ability of the lens to repair itself, as found in a previous single dose study, was absent after repeated doses. Lenses exposed daily to 0.03 and 0.01 J/cm2 UV-B showed no significant change in transmittance or focal length variability compared to controls. Daily exposure to 1.44 J/cm2 UV-A resulted in no significant change in transmittance or focal length variability compared to controls.

Cold cataracts were induced in ten bovine lenses and then removed by warming. Cataracts first appeared at an average temperature of 11.7 degrees C. The cataracts appeared to be densest at an average temperature of 1.2 degrees C, while warming caused them to disappear completely at an average temperature of 16.4 degrees C. A computer-operated scanning laser system was used to measure the equivalent focal length and changes in relative transmittance before, during, and after the cataract was induced. In general the focal length profile (spherical aberration) that existed before cooling was recaptured on warming. Scatter values indicate that transmittance is not affected by the temporary cold cataract. Thus the optical performance of the bovine lens appears to be identical before and after cold cataracts are induced. We believe that these results indicate that the cataract has a supramolecular origin.

Young rainbow trout were given diets containing graded levels of methionine for 16 wk. Analysis of the weight gain and food efficiency data showed the methionine requirement to be not more than 0.76% of the diet (1.9% of dietary protein). Activities of regulatory enzymes of the transulfuration pathway, methionine adenosyltransferase and cystathionine synthase in trout liver were not altered by changes in methionine intake. Concentrations of free serine in liver and plasma of the trout were high at low levels of methionine intake but fell as dietary methionine increased. This implied decreased flux through cystathionine synthase at low methionine intakes. Large increases in liver and plasma taurine occurred at high methionine intakes, implying enhanced transulfuration activity. Liver ornithine decarboxylase activity was reduced at the lowest level of dietary methionine used but the activity of S-adenosylmethionine decarboxylase was unchanged. Eye lenses of the trout given these diets were examined by a scanning lens monitor. Analysis of focal length variability with this equipment demonstrated that, if abnormality of the lens is to be avoided, a higher concentration of dietary methionine (0.96% or 0.6% methionine + 0.36% cystine) is needed than that required to maximize growth.

The effect of UV-B radiation on the crystalline lens was examined by subjecting bovine lenses in culture to varying low exposures at 300 nm. Lens optical quality was monitored on a long-term basis (to 1000 hrs.) with an automated scanning laser system that recorded both change in relative scatter and focal length across each lens. Data were collected for 20 lens positions at each scan. Radiant exposure levels consisted of 0.5, 0.25, 0.125, 0.06 and 0.03 Jcm-2. Twenty irradiated lenses were compared to twelve untreated controls. All of the irradiated lenses showed changes in scatter and focal length relative to the controls. Most (about 75%) of the treated lenses showed significant increases in scatter (200-400%) and focal length (10-20%) at 40 to 60 hours after exposure. A similar time frame for lens damage was noted by visual inspection. Exposure to UV-B at the above doses did not affect culture longevity.

Transcatheter embolization by Ivalon particles for treatment of arteriovenous malformations has been an accepted therapeutic technique for many years. We describe a new and efficient radiolabeling technique of Ivalon particles using [99mTc]sulfur colloid. Continuous and dynamic monitoring of injected radiolabeled Ivalon particles is made possible by viewing the persistence scope of a portable gamma camera whose head is positioned over the patient undergoing therapeutic embolization. Therefore, if inadvertent pulmonary embolism or reflux migration of radiolabeled Ivalon particles has occurred, the angiographer is immediately aware of this potentially serious or fatal complication and can take corrective action. We describe two patients, each with an arteriovenous malformation, who had therapeutic embolization with radiolabeled Ivalon particles, one resulting in reflux migration and the other resulting in inadvertent pulmonary embolism.